Download Purified Hexameric Epstein-Barr Virus-Encoded BARF1 Protein for Measuring Anti-BARF1 Antibody

Purified Hexameric Epstein-Barr
Virus-Encoded BARF1 Protein for
Measuring Anti-BARF1 Antibody
Responses in Nasopharyngeal Carcinoma
Patients
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E. K. Hoebe, S. H. Hutajulu, J. van Beek, S. J. Stevens, D. K.
Paramita, A. E. Greijer and J. M. Middeldorp
Clin. Vaccine. Immunol. 2011, 18(2):298. DOI:
10.1128/CVI.00193-10.
Published Ahead of Print 1 December 2010.
CLINICAL AND VACCINE IMMUNOLOGY, Feb. 2011, p. 298–304
1556-6811/11/$12.00 doi:10.1128/CVI.00193-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 18, No. 2
Purified Hexameric Epstein-Barr Virus-Encoded BARF1 Protein for
Measuring Anti-BARF1 Antibody Responses in Nasopharyngeal
Carcinoma Patients䌤
E. K. Hoebe,1 S. H. Hutajulu,2 J. van Beek,1† S. J. Stevens,1‡ D. K. Paramita,2
A. E. Greijer,1 and J. M. Middeldorp1*
Received 6 May 2010/Returned for modification 28 June 2010/Accepted 11 November 2010
WHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with
Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is
considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in
the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1
and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric
BARF1 in sera of Indonesian NPC patients (n ⴝ 155) compared to healthy EBV-positive (n ⴝ 56) and
EBV-negative (n ⴝ 16) individuals. BARF1 (B95-8) expressed in Escherichia coli and baculovirus, as well as
BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1
protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed
relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV
proteins. Higher IgG reactivity was found in NPC patients (P ⴝ 0.015) than in regional Indonesian controls
or EBV-negative individuals (P < 0.001). IgA responses to native BARF1 were marginal. NPC sera with the
highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by
immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native
conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC
patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.
ing on the viral lytic cycle (11, 27). Direct demonstration of
BARF1 protein expression in carcinoma tissue has proven
extremely difficult, although one report described its presence
in NPC tumor extracts (5). Recently, it was shown that BARF1
lacking the first 20 amino acids is actively secreted (6, 7, 30),
and BARF1 protein was detected in sera of NPC patients in
amounts of 500 to 5,000 ng/ml, but not in healthy EBV carriers
(13).
The functions assigned to BARF1 are diverse. BARF1 has
been shown to have transforming activity and to prevent senescence (33, 44, 45) and apoptosis (3, 42). Secreted BARF1
protein (sBARF1) has been reported to have mitogenic activity
on human B cells and primary monkey kidney epithelial cells
(30). A possible role for sBARF1 as an immune-modulating
protein was suggested, since Fc-tagged BARF1 protein was
able to act as an antagonist for macrophage colony-stimulating
factor (M-CSF) (4, 36). Parts of the BARF1 protein are homologous to the Ig superfamily of receptors, and a small domain has homology with the T cell receptor costimulatory
molecule CD80 (4, 36, 38). However, the exact function of the
secreted BARF1 protein in EBV-related carcinoma is still under investigation.
Nasopharyngeal carcinomas are characterized by a significant infiltrate of CD4⫹ and CD8⫹ T cells (15). Therefore,
BARF1 is expected to trigger immune responses. Indeed, T
cell responses against BARF1-derived peptides were recently
detected in NPC patients, opening options for immune therapy
(18). However, lymphocytes obtained from the NPC tumor
environment are functionally impaired (17), suggesting local
immune modulation, which may be linked to BARF1. Anti-
Epstein-Barr virus (EBV) is a human gammaherpesvirus
with tropism for B lymphocytes and epithelial cells. EBV infection occurs worldwide, and about 90% of the world population is persistently infected. EBV is etiologically linked to
several lymphoid and epithelial malignancies, with the latter
including most undifferentiated and poorly differentiated nasopharyngeal carcinomas (NPC) (WHO types II and III, respectively) (16, 29) and about 10% of gastric adenocarcinomas
(GC) worldwide (22, 32, 47). NPC has a well-defined geographical distribution and is particularly prevalent in Southeast
Asia. Both genetic and dietary influences are thought to be
important in NPC etiology (2, 48). NPC shows latency type II
EBV transcription in all tumor cells, with expression of the
noncoding small RNAs EBER1 and -2 (EBER1/2), BamHI A
rightward transcripts (BARTs), and Epstein-Barr nuclear antigen 1 (EBNA1) (10, 46). Latent membrane protein 1 (LMP1)
and LMP2 are more heterogeneously expressed (1, 10, 46, 49).
Furthermore, transcription of an additional viral gene in
BamHI-A rightward frame 1 (BARF1) was described previously (5, 34, 35, 50). BARF1 mRNA is exclusively expressed in
EBV-positive carcinomas and is absent from EBV-positive
lymphomas (12, 32, 43). However, it can be activated by switch* Corresponding author. Mailing address: Department of Pathology,
VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, Netherlands. Phone: 31-20-4442168. Fax: 31-20-4442964. E mail:
E-mail: [email protected].
† Present address: RIVM, Bilthoven, Netherlands.
‡ Present address: Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, Netherlands.
䌤
Published ahead of print on 1 December 2010.
298
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VU University Medical Center, Department of Pathology, Amsterdam, Netherlands,1 and Gadjah Mada University, Yogyakarta, Indonesia2
VOL. 18, 2011
IMMUNE RESPONSES TO NATIVE EBV PROTEIN BARF1
MATERIALS AND METHODS
Sera from NPC patients and healthy EBV carriers. Serum panels from histologically confirmed NPC patients (n ⫽ 155) were collected at the Department of
Ear, Nose, and Throat (ENT), Dr. Sardjito General Hospital, Yogyakarta, Indonesia. NPC sera were taken prior to treatment. NPC staging was done by ENT
examination and computed tomography (CT) scan and classified according to the
1996 Union International Cancer Control (UICC) classification. Sera from EBVpositive healthy individuals (n ⫽ 56) were obtained from the Indonesian Red
Cross blood bank, Yogyakarta, Indonesia, and the archives of the VU University
Medical Center Department of Pathology, respectively, and have been extensively characterized (19, 20, 28, 39). EBV-negative sera (n ⫽ 16) were collected
from healthy individuals in the Netherlands.
Plasmids. The B95-8 prototype BARF1 sequence was obtained from JY cells
by PCR and cloned into the pCRScript vector (Stratagene, Agilent Technologies,
Amstelveen, Netherlands). Subsequently, the sBARF1 B95-8 sequence (amino
acid [aa] 21 to aa 221) was cloned into the pHTA donor vector, adding a 6-His
tag C terminally (BARF1-His vector). In addition, the sBARF1 sequence (aa 21
to aa 221) was cloned into the pGEX 4T-3 vector (Amersham Biosciences, GE
Healthcare, Diegem, Belgium), adding a glutathione S-transferase (GST) tag
(BARF1-GST vector). A consensus BARF1 DNA sequence from an Indonesian
NPC patient was obtained from S. Hutajulu (14). This BARF1 sequence differs
from B95-8 at positions 29 (valine to alanine) and 130 (histidine to arginine) and
was used for construction of the BARF1 expression vector pcDNA-BARF1. The
amplification product was cloned into a pcDNA4/TO vector (Invitrogen, Breda,
Netherlands) (referred to below as native sBARF1).
Cell culture. 293HEK cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM); an EBV-negative NPC-derived cell line, Hone1, was cultured
in RPMI medium (Lonza, Breda, Netherlands); and the gastric cancer cell line
AGS was cultured in HAM’s nutrient mixture F12 (Sigma-Aldrich, Zwijndrecht,
Netherlands) at 37°C and 5% CO2 in a humidified atmosphere. All media
contained 10% fetal calf serum (FCS), 100 units/ml penicillin (PEN), 100 ␮g/ml
streptomycin (STR), 2 mM L-glutamine, and 50 ␮M ␤-mercaptoethanol. After
stable transfection with pcDNA-BARF1, 293HEK-BARF1 cells were cultured
under selection with 95 ␮g/ml phleomycin (Zeocin). Spodoptera frugiperda (Sf9)
insect cells were cultured in Sf-900 II SFM with L-glutamine medium (Invitrogen) at 27°C.
BARF1 protein expression and purification. Recombinant baculovirus
(BARF1-His vector) was produced as described previously (21). BARF1-GST
was generated by inducing pGEX-BARF1-transformed Escherichia coli with 10
mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 4 h and purified with glutathione-Sepharose (Pharmacia, GE Healthcare) as described by the manufacturer. NPC-derived human-secreted BARF1 (native-sBARF1) protein was produced by stable transfection of 293HEK cells with the pcDNA-BARF1 vector
using Lipofectamine (Invitrogen). For protein production, cells were washed
twice with phosphate-buffered saline (PBS), and serum-free medium was added
for 24 h, after which the medium was harvested. Medium (300 ml) was diluted 1:1
in binding buffer (20 mM Tris, pH 7.4, 0.5 M NaCl) and incubated for 1 h with
10 ml concanavalin A bead slurry (Sigma-Aldrich). After being washed, BARF1
was eluted with 500 mM methyl-alpha-D-glucopyranoside in binding buffer. Mul-
tiple elutions were concentrated to 1 ml with a Centriprep 50 device (Millipore,
Amsterdam, Netherlands) and loaded onto a 25-ml Superdex-200 Sepharose size
exclusion column (Pharmacia, GE Healthcare). Fractions containing sBARF1
were pooled and dialyzed against PBS overnight at ⫹4°C. The protein concentration was determined by using a BCA Protein Assay kit (Pierce, Thermo Fisher
Scientific, Etten Leur, Netherlands).
Synthetic peptides. Antigenic peptide epitopes of sBARF1 were derived by
computer prediction techniques, as described previously (24, 25, 40), using high
scores for hydrophilicity, flexibility, and ␤-turn probability. Peptides named
BARF1 N peptide (aa 40 to 79), M peptide (aa 151 to 188), and C peptide (aa
187 to 221) (Fig. 1A) were synthesized with a peptide synthesizer (433A; Applied
Biosystems, Nieuwerkerk a/d IJssel, Netherlands) using 9-fluorenylmethoxy carbonyl (Fmoc) amino acids purchased from Bachem. Peptides were purified by
reverse-phase high-performance liquid chromatography.
Monoclonal and polyclonal antibodies. Monoclonal antibodies (MAb) 4A6
and 6F4 were produced by immunization of mice with synthetic BARF1 C
peptide. Polyclonal antibody (PAb) K150.3 was produced by immunization of
rabbits with purified recombinant BARF1-His protein and administered in
Freund’s adjuvant. Guinea pig antibodies specific for defined domains of BARF1
(GP␣C, GP␣M, and GP␣N) were prepared by immunizing animals with synthetic BARF1 peptides coupled with keyhole limpet hemocyanin and administered in Freund’s adjuvant. Rabbit polyclonal antibodies to native BARF1 (PAb
nativeBARF1) was produced by immunization of a rabbit with purified native
sBARF1 from 293HEK-BARF1 cells and administered in Immacel-R adjuvant
(Pickcell Laboratories BV, Amsterdam, Netherlands).
SDS-PAGE and Western blot analysis. Cell samples were lysed in PBS containing 1% Triton X-100 and protease inhibitor cocktail (Roche, Almere, Netherlands) and sonified. Cell debris was removed by centrifugation, and the protein
concentration was determined using a BCA Protein Assay kit. For sBARF1, the
medium was harvested, and debris was removed by centrifugation. For reduced
SDS-PAGE, samples were diluted in 2⫻ loading buffer (Bio-Rad, Veenendaal,
Netherlands) with ␤-mercaptoethanol and heated for 5 min at 95°C. For nonreduced SDS-PAGE, samples were diluted in 2⫻ nonreducing loading buffer
(Bio-Rad) and incubated for 10 min at room temperature (RT). The samples
were run on a 12.5% SDS-PAGE gel and either transferred to a Hybond ECL
nitrocellulose membrane (GE Healthcare) or Coomassie stained with Instant
Blue (Expedeon, Westburg, Leusden, Netherlands). After being blotted, the
membrane was blocked in PBS containing 3% nonfat dry milk for 1 h at RT.
Incubation with 4A6 primary antibody (1:100) in PBS containing 0.05% Tween
20 (PBST) with 5% bovine serum albumin (BSA) was followed by IRDye800CWconjugated secondary antibodies (1:1,000) (Li-Cor, Westburg, Netherlands) in
PBST containing 3% milk. Nitrocellulose blots were visualized using the Odyssey
Infrared Imaging System (Li-Cor).
Glycosylation and phosphorylation analysis. Denaturing buffer (1 ␮l) was
added to 9 ␮l of sBARF1-containing medium and placed at 95°C for 10 min.
After cooling, PNGase-F (NEB, Westburg, Netherlands), O-glycosidase, or
neuraminidase (Sigma-Aldrich) and matching buffer were added and incubated
for 90 min at 37°C. For blocking BARF1 secretion, cells were exposed for 24 h
to either 3 ␮g/ml brefeldin A or 50 ␮M monensin (Sigma-Aldrich). Phosphorylation of BARF1 was analyzed by immunoblotting using anti-phosphoserine
(1:200; Sigma-Aldrich) and anti-phosphothreonine (1:1,000; Cell Signaling Technology, Bioké, Leiden, Netherlands) antibodies.
EBV immunoblotting. Viral capsid antigen (VCA) was produced in HH514,
and EBV-VCA immunoblot strips were made as described previously (23). For
detection of EBV antibodies, blot strips were incubated with human sera (1:100).
After 3 washes with PBST, the secondary antibody rabbit anti-human IgG (1:
1,000; Dako, Glostrup, Denmark) was added. After three washing steps in PBST
followed by PBS, bands were visualized either using 0.06% (wt/vol) 4-chloronaphthol (Sigma-Aldrich) and 0.01% (vol/vol) H2O2 in PBS or with ECL⫹
detection reagent (Amersham).
Anti-BARF1 ELISA. Individual wells of Costar-9018 high-binding 96-well enzyme-linked immunosorbent assay (ELISA) plates (Corning, VWR, Amsterdam,
Netherlands) were coated with 1 ␮g/ml BARF1 peptide or 10 ␮g/ml purified
sBARF1 protein in 0.05 M NaHCO3, pH 9.6, overnight at 4°C, followed by
blocking with 3% BSA in PBS for 1 h at 37°C. Serum samples were tested in
duplicate; diluted 1:100 in PBS containing 0.05% Tween, 0.1% Triton X-100, 1%
BSA (PBS-TTB); and incubated for 1 h at 37°C. After 4 washes with PBST, the
plate was incubated with rabbit anti-human IgG (1:8,000) or IgA (1:4,000) in
PBS-TTB and incubated for 1 h at 37°C. After 4 washes with PBST, color was
developed using TMB substrate (bioMérieux, Boxtel, Netherlands), and the
optical density at 450 nm (OD450) was measured.
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body-dependent cellular cytotoxicity against BARF1-transfected Raji cells using sera of NPC patients has been described
(37). Unfortunately, the study does not agree with the more
recent knowledge of rapid and complete secretion of BARF1.
Aberrant EBV serology is commonly used to support NPC
diagnosis and provides an affordable approach for population
screening (8, 26). Antibodies targeting the BARF1 protein
could be used as a new diagnostic tool to identify NPC patients.
In this study, we analyze the humoral immune response to
BARF1 protein in a large group of Indonesian NPC patients.
Here, we employ a BARF1 sequence found in NPC with 2
conservative mutations, V29A and H130R, which are not considered to affect the three-dimensional (3D) structure significantly (14). This protein was expressed in human 293 cells and
purified as a hexameric protein. The use of “native” hexameric
BARF1 protein proved to be essential in detecting antiBARF1 antibody responses in NPC patients.
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Data analysis. The Mann-Whitney test was performed with the SPSS statistical
program, and P values under 0.05 were considered significant. SPSS was also
used to calculate the Pearson correlation coefficient.
RESULTS
Lack of antibody response against nonnative B95-8-derived
recombinant BARF1 protein and BARF1 peptides. Since
BARF1 mRNA and BARF1 protein are abundantly expressed
in NPC patients (35, 50), antibody reactivity to BARF1 was
evaluated as a potential new diagnostic marker. In a first approach to study BARF1-specific antibody reactivity in NPC
patients by ELISA, BARF1-derived peptides with predicted
antigenic domains were selected (Fig. 1A). Antibodies were
generated against the three selected synthetic peptides, as well
as against a purified full-length baculovirus-encoded BARF1His fusion protein expressed in insect cells. The antibodies
reacted specifically in ELISA to their respective peptides (Fig.
1B), and all reacted with extracts of BARF1-expressing insect
cells by immunocytochemical staining and immunoblot analysis (data not shown). Antibodies raised against full-length
BARF1-His fusion protein displayed a stronger reaction to the
C peptide. However, none of the human sera displayed reactivity to any of the 3 peptides used, despite having abundant
antibody reactivity with a wide range of EBV proteins and
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FIG. 1. BARF1 (peptide) sequence and specific animal antibody reactivity against B95-8-derived recombinant BARF1 protein and antigenic
peptides. (A) BARF1 sequence containing 2 common amino acid substitutions, V29A and H130R, found in Indonesian isolates (boldface).
Antigenic peptides designed by computer analysis, named N peptide (aa 40 to 79), M peptide (aa 151 to 188), and C peptide (aa 187 to 221), are
underlined. (B) Antibody reactivity to peptides measured in ELISA. Guinea pig antibodies against C peptide, M peptide, N peptide, and mouse
monoclonal anti-C peptide (4A6) specifically react to their target peptides. The rabbit polyclonal antibody anti-BARF1-His (K150.3) binds to both
C and M peptides. Rabbit polyclonal antibody raised against the purified native sBARF1 reacts to the C peptide. (C) Antibody reactivity of healthy
individuals and NPC patients to the BARF1 C peptide measured in IgG ELISA, showing slightly elevated levels in NPC patients. (D) IgG
immunoblot analysis of 3 representative NPC sera with BARF1-His expressed by the Sf9 insect cell expression system and BARF1-GST expressed
by E. coli. HH514-derived viral capsid antigens were used as a positive control for EBV antibody presence in patient sera. Guinea pig anti-C
peptide antibody (⫹) showed a clear band on the blot strips, but no BARF1 reactivity was detected in the patient sera despite the presence of
diverse antibody responses to other EBV proteins, as visualized on the HH514 strips.
VOL. 18, 2011
IMMUNE RESPONSES TO NATIVE EBV PROTEIN BARF1
FIG. 2. BARF1 is secreted as a hexameric glycosylated protein
from human HEK293 cells. (A) BARF1 is secreted as a hexamer in the
culture supernatant of stably transfected 293HEK-BARF1 cells. Ten
microliters of medium was mixed 1:1 with either reduced (R) or nonreduced (NR) loading buffer and separated by SDS-PAGE and Western blotting. A 29-kDa and a 160-kDa band can be seen, representing
the monomeric and the hexameric structures of sBARF1, respectively.
(B) The glycosylation status of BARF1 in serum-free culture supernatant was determined by direct treatment with several glycosidases.
PNGase F cleaves high-mannose N-linked sugar groups. Neuraminidase removes sialic acid groups, and O-glycosidase cleaves O-linked
sugar chains. The digested protein was loaded on an SDS-PAGE
Western blot and detected using 4A6 anti-BARF1 antibody. (C) Manipulation of BARF1 secretion from HEK293 cells. Under normal
growth conditions, hardly any BARF1 can be detected in 293HEK cell
lysates; BARF1 is completely secreted in the culture supernatant.
Treatment of cells with the transport blockers monensin (Mon) and
brefeldin A (BFA) retains sBARF1 in the cell lysate and almost completely blocks secretion into the culture medium. (D) Schematic overview of intercellular transport blockage by brefeldin A and monensin.
(E) Native-sBARF1 was purified from culture medium using ConA
affinity chromatography followed by size exclusion chromatography
and visualized by Coomassie staining as hexameric sBARF1 (NR) and
monomeric sBARF1 (R). Purity of ⬃98% was routinely reached in
repeated experiments.
therefore, hardly any BARF1 was detected in 293 cell lysate
(Fig. 2C). To determine whether BARF1 is indeed secreted via
the classical pathway, specific blockers were used. Blockage of
BARF1 protein passage at specific stages in the secretory pathway was performed with brefeldin A, which disturbs the Golgi
apparatus, and monensin, which halts glycoproteins in the endoplasmic reticulum (ER) (Fig. 2D). Both reagents prevented
BARF1 secretion into the medium and allowed protein accumulation inside BARF1-expressing cells (Fig. 2C). Furthermore, brefeldin A and monensin resulted in BARF1 bands at
lower molecular weight than BARF1 secreted in medium (Fig.
2C), which can be explained by reduced glycosylation status.
Low antibody responses against native BARF1 exist in NPC
patients. HEK293-produced native sBARF1 protein contain-
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EBV peptide sequences (8, 9, 28). Only BARF1 C peptide
generated a minor IgG response in 9.5% (4/42) of the NPC
patient sera (Fig. 1C).
In order to investigate whether human antibodies might be
reactive against other determinants on BARF1, purified fulllength BARF1-GST (E. coli) and BARF1-His (insect cells)
were used in immunoblot analysis to probe for reactivity in sera
of NPC patients (n ⫽ 149). EBV reactivity of sera on immunoblot strips with EBV VCA protein extracts revealed antibody responses to multiple EBV proteins, i.e., EBNA1, EAdTK, EAd-p47/54, ZEBRA, and VCA-p18, as shown in Fig. 1D
(9). Whereas BARF1-specific polyclonal control antibodies
gave a clear staining pattern on the strips, no specific antiBARF1 response was observed in any of the human serum
samples analyzed using either Sf9 insect cell BARF1-His, E.
coli-produced BARF1-GST, or the selected peptide antigens.
BARF1 peptides and BARF1-His or BARF1-GST proteins
produced in nonhuman expression systems may not have the
proper folding and conformation. Therefore, we subsequently
focused on the use of an NPC-derived BARF1 sequence produced in a human cell background.
NPC-derived sBARF1 is secreted as a glycosylated hexameric protein from human cells. Sequence analysis of the
BARF1 gene revealed two prevalent amino acid substitutions
(V29A and H130R) in Indonesian NPC patients, which were
predicted to be conservative in nature, leading to only minor
changes in protein folding (14). A stable NPC-derived BARF1expressing 293HEK cell line was created. The expressed
BARF1 protein was rapidly and completely secreted into the
culture medium and was detected as a 27- to 29-kDa band
under denaturing conditions by SDS-PAGE and Western blot
analysis (Fig. 2A), in agreement with previously reported data
(7, 31). Under nonreducing conditions, sBARF1 produced by
293HEK cells revealed a 160- to 180-kDa molecule representing the hexameric form of BARF1 (Fig. 2A). Similar to the
293HEK system, BARF1 expressed transiently in Hone1
(NPC) and AGS (gastric carcinoma) cells was rapidly and
completely secreted from the cells (data not shown). Earlier,
BARF1 was described as a glycosylated protein (7, 38). To
analyze the glycosylation status of secreted BARF1 protein
(sBARF1), selective glycosidase analysis was performed. Specific enzymes were used to remove O-linked sugar chains,
N-linked sugar chains, and sialic end groups of sBARF1 protein. PNGase F cleavage created a major band shift (Fig. 2B),
indicating that sBARF1 contained high-mannose N-linked glycosylation. Neuraminidase was used to cut off sialic end groups
from sugars, which resulted in a minor band shift (Fig. 2B).
O-Glycosidase can cut off a sugar group only when incubated
together with neuraminidase. This combination demonstrates
the presence of sialic acid end groups, as well as O-linked
glycosylation, in the BARF1 protein (Fig. 2B). We conclude
that HEK293-expressed sBARF1 is an N-linked glycosylated
protein with additional O-linked glycosylic modifications carrying sialic end groups.
Phosphorylation of secreted BARF1 protein was examined
with anti-phosphothreonine and anti-phosphoserine monoclonal antibodies. Although a band was detected with BARF1specific antibody (4A6), no band was observed with phosphospecific antibodies (data not shown).
BARF1 protein is rapidly and completely secreted, and
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ing proper glycosylation and hexameric folding was purified
with concanavalin A (ConA) and size exclusion chromatography and used to develop an antibody capture ELISA. The
purity of the hexameric native sBARF1 was over 98%, as was
shown by Coomassie staining (Fig. 2E). Pure native sBARF1
protein was used to develop an antibody capture ELISA. Optimization was performed with anti-BARF1 antibodies and
controls (preimmune sera and non-EBV MAbs, as shown in
Fig. 1B). Subsequently, sera from Indonesian NPC patients
(n ⫽ 155) and healthy EBV-positive (n ⫽ 55) and EBV-negative (n ⫽ 16) Caucasian and Indonesian individuals were
tested. A weak but clear IgG response against native sBARF1
was observed in NPC patients, which showed significantly
higher reactivity than regional controls (P ⫽ 0.015) and EBV-
negative individuals (P ⬍ 0.001) (Fig. 3A). Healthy Indonesian
and Dutch EBV carriers had similar responses. To analyze
whether anti-BARF1 responses were related to the level of
humoral immunity to other immunodominant EBV proteins,
we analyzed anti-VCA and anti-EBNA1 antibody responses in
individual NPC patients. The results shown in Fig. 3B and C
reveal no correlation between these responses, indicating that
anti-BARF1 antibodies are formed independently and at a
relatively low level. Although in many NPC patients antiBARF1 IgA was barely detectable, overall, NPC patients had
significantly increased IgA levels against sBARF1 compared to
the controls, albeit less pronounced than IgG responses (Fig.
3A and D). Healthy EBV carriers were virtually devoid of
anti-BARF1 responses, as shown in Fig. 3A. The proportion of
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FIG. 3. Low immune responses against native BARF1 in human sera. (A) IgG antibodies against native human sBARF1 detected by antibody
capture ELISA. Data are shown as OD450 values for EBV-negative healthy donors (n ⫽ 16), EBV-positive healthy donors (n ⫽ 56), and NPC
patients (n ⫽ 155). (B) IgA antibodies against native human sBARF1 were detected by ELISA using EBV-negative healthy donors (n ⫽ 15),
EBV-positive healthy donors (n ⫽ 57), and NPC patients (n ⫽ 155). (C) Scatter plot of IgG anti-BARF1 versus IgG anti-EBNA responses.
(D) Scatter plot of IgG anti-BARF1 versus IgG anti-VCAp18 responses. Filled circles, NPC patients; open circles, EBV-positive healthy donors;
stars, EBV-negative healthy donors.
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303
patients with positive IgG-BARF1 test results who were correctly diagnosed with NPC by pathological examination (positive predictive value) was 100%, but due to the limited immunogenicity of BARF1 in vivo, many true NPC patients lack
detectable anti-BARF1 responses, thus yielding a negative predictive value of 28%.
The specificity of the response against native sBARF1 was
analyzed by using immunoblot analysis. Immunoblot strips
containing purified sBARF1 were incubated with sera from the
strongest BARF1 responders in ELISA. A thin BARF1 band
was visible for some sera. Only with considerable overexposure
was a BARF1 band visualized with most of the sera (Fig. 4),
confirming the low-immunogenic nature of BARF1 protein.
The staining intensities of the sera on immunoblots did not
correlate with OD450 values in antibody capture ELISA, suggesting alternative epitopes may be detected by both techniques (data not shown).
DISCUSSION
Since transcription of the EBV BARF1 gene was found to
be selectively associated with EBV-related malignancies of epithelial origin, substantial research has been done to determine
its function and potential role in EBV-driven carcinogenesis.
Recently, BARF1 was considered a diagnostic marker, since it
was reported to be actively secreted from BARF1-expressing
cells in model systems (6, 7). Secreted BARF1 protein was also
detected in sera of NPC patients (13). However, the abundance of BARF1 protein in patient sera, as suggested by
Houali et al., remains to be confirmed. The expression and
secretion of BARF1 in human carcinoma suggest that BARF1
might trigger antibody responses. Using NPC sequence
sBARF1 from a human expression system, we showed in this
study the presence of anti-BARF1 antibodies in NPC patients
and healthy EBV-positive individuals. Despite the use of native NPC-derived sBARF1, the anti-BARF1 antibody response
remained low.
Initially, a recombinant B95-8 sequence-derived BARF1 fusion protein expressed in bacterial and insect cell systems did
not reveal any reactivity with human sera. Even in a sensitive
ELISA, predicted antigenic peptides did not yield detectable
anti-BARF1 responses despite excellent results for other EBV
markers in our prior work (8, 24, 40, 41). Peptide C is the only
BARF1 domain that appeared to generate a minor humoral
immune response, as well as for the developed BARF1 monoclonal and polyclonal antibodies. These results favor the C
terminus of BARF1 as the most immunogenic BARF1 domain.
Recently, Tarbouriech et al. provided direct evidence that
the native form of secreted BARF1 was a glycosylated hexameric protein (38). Since insect cells and bacteria differ from
human cells in folding and posttranslational modifications, the
need arose for a human expression system of recombinant
BARF1 protein, better reflecting the natural form. Sequence
analysis of the EBV BARF1 gene of NPC patients revealed
two amino acid changes compared to the generally used B95-8
BARF1 sequence (14). These amino acid changes are located
at surface-accessible sites on the native BARF1 protein and
are suggested to yield only marginal changes in BARF1 protein
folding compared to the B95-8 prototype but may affect antibody binding to native BARF1 (14, 38). This NPC-based
BARF1 sequence was used for producing a human recombinant BARF1 (HEK293-BARF1). HEK293-BARF1 cells rapidly secrete the sBARF1 protein in the culture medium as a
hexameric protein (6, 32). The secreted BARF1 protein is
N-linked and O-linked glycosylated, the latter also containing
sialic acid modification at the termini. We were not able to
confirm phosphorylation of sBARF1 protein, as was suggested
previously (6).
Using purified native sBARF1 protein in an antibody capture ELISA, we detected IgG antibody responses against
BARF1 in some healthy EBV carriers and most NPC patients. NPC patients responded significantly more strongly
to BARF1 than healthy controls (P ⫽ 0.015). The presence
of a weak response in EBV carriers can be explained by
transcription of BARF1 during the lytic cycle in normal
EBV infection. The IgA response to sBARF1 was low and
difficult to detect.
Our data reveal BARF1 as a marginally immunogenic
protein in patients with otherwise strong responses to alternative EBV proteins, such as VCA-p18, EBNA1, and Zebra.
The observation that anti-BARF1 antibodies were mainly
directed against conformational epitopes on purified native
BARF1 protein suggests that BARF1 may circulate in vivo
as a free hexameric protein, possibly interacting with M-CSF
to modulate immune responses, an interaction that is only
minimally interfered with by humoral immune responses.
Downloaded from http://cvi.asm.org/ on December 9, 2011 by VRIJE UNIVERSITEIT
FIG. 4. Immunoblot confirmation of specific antibody responses to native sBARF1. Native sBARF1 was loaded on a reduced SDS-PAGE gel,
blotted to nitrocellulose, and cut into strips. Sera from the highest responders in the NPC patient panel and control sera were analyzed at 1:100
dilution. 4A6 anti-BARF1 antibody was used as a positive control (C). A band at the correct height was detected with most of the selected NPC
patient sera only upon considerable overexposure (minutes) when using ECL visualization. The 4A6 signal was revealed in a few seconds.
304
HOEBE ET AL.
CLIN. VACCINE IMMUNOL.
ACKNOWLEDGMENTS
This project was financially supported by the Dutch Cancer Society
Project VU2007-3776 and the Nuffic/Netherlands Fellowship Program
(NFP-PhD 08/36).
We are grateful to Liesbeth Cairo, Floortje Krechting, and Bo van
Kuijk for their technical assistance.
We declare no conflict of interest.
25.
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REFERENCES
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
Downloaded from http://cvi.asm.org/ on December 9, 2011 by VRIJE UNIVERSITEIT
1. Brooks, L., Q. Y. Yao, A. B. Rickinson, and L. S. Young. 1992. Epstein-Barr
virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J. Virol. 66:2689–2697.
2. Chang, E. T., and H. O. Adami. 2006. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol. Biomarkers Prev. 15:1765–1777.
3. Chang, M. S., et al. 2005. Cell-cycle regulators, bcl-2 and NF-kappaB in
Epstein-Barr virus-positive gastric carcinomas. Int. J. Oncol. 27:1265–1272.
4. Cohen, J. I., and K. Lekstrom. 1999. Epstein-Barr virus BARF1 protein is
dispensable for B-cell transformation and inhibits alpha interferon secretion
from mononuclear cells. J. Virol. 73:7627–7632.
5. Decaussin, G., F. Sbih-Lammali, M. de Turenne-Tessier, A. Bouguermouh,
and T. Ooka. 2000. Expression of BARF1 gene encoded by Epstein-Barr
virus in nasopharyngeal carcinoma biopsies. Cancer Res. 60:5584–5588.
6. de Turenne-Tessier, M., P. Jolicoeur, and T. Ooka. 1997. Expression of the
protein encoded by Epstein-Barr virus (EBV) BARF1 open reading frame
from a recombinant adenovirus system. Virus Res. 52:73–85.
7. de Turenne-Tessier, M., and T. Ooka. 2007. Post-translational modifications
of Epstein Barr virus BARF1 oncogene-encoded polypeptide. J. Gen. Virol.
88:2656–2661.
8. Fachiroh, J., et al. 2006. Single-assay combination of Epstein-Barr virus
(EBV) E. J. Clin. Microbiol. 44:1459–1467.
9. Fachiroh, J., et al. 2004. Molecular diversity of Epstein-Barr virus IgG and
IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J. Infect. Dis. 190:53–62.
10. Fåhraeus, R., et al. 1988. Expression of Epstein-Barr virus-encoded proteins
in nasopharyngeal carcinoma. Int. J. Cancer 42:329–338.
11. Hatfull, G., A. T. Bankier, B. G. Barrell, and P. J. Farrell. 1988. Sequence
analysis of Raji Epstein-Barr virus DNA. Virology 164:334–340.
12. Hayes, D. P., et al. 1999. Expression of Epstein-Barr virus (EBV) transcripts
encoding homologues to important human proteins in diverse EBV associated diseases. Mol. Pathol. 52:97–103.
13. Houali, K., et al. 2007. A new diagnostic marker for secreted Epstein-Barr
virus encoded LMP1 and BARF1 oncoproteins in the serum and saliva of
patients with nasopharyngeal carcinoma. Clin. Cancer Res. 13:4993–5000.
14. Hutajulu, S. H., et al. 2010. Conserved mutation of Epstein-Barr virusencoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma. Infect. Agents Cancer 5:16.
15. Jayasurya, A., B. H. Bay, W. M. Yap, and N. G. Tan. 2000. Lymphocytic
infiltration in undifferentiated nasopharyngeal cancer. Arch. Otolaryngol.
Head Neck Surg. 126:1329–1332.
16. Klein, G., et al. 1974. Direct evidence for the presence of Epstein-Barr virus
DNA and nuclear antigen in malignant epithelial cells from patients with
poorly differentiated carcinoma of the nasopharynx. Proc. Natl. Acad. Sci.
U. S. A. 71:4737–4741.
17. Li, J., et al. 2007. Functional inactivation of EBV-specific T-lymphocytes in
nasopharyngeal carcinoma: implications for tumor immunotherapy. PLoS
One 2:e1122.
18. Martorelli, D., et al. 2008. Spontaneous T cell responses to Epstein-Barr
virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy. Int. J. Cancer
123:1100–1107.
19. Meij, P., et al. 2002. Identification and prevalence of CD8(⫹) T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2. Int. J. Cancer 99:93–99.
20. Meij, P., et al. 1999. Restricted low-level human antibody responses against
Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup
of patients with EBV-associated diseases. J. Infect. Dis. 179:1108–1115.
21. Meij, P., M. B. Vervoort, C. J. Meijer, E. Bloemena, and J. M. Middeldorp.
2000. Production monitoring and purification of EBV encoded latent membrane protein 1 expressed and secreted by recombinant baculovirus infected
insect cells. J. Virol. Methods 90:193–204.
22. Middeldorp, J. M., A. A. Brink, A. J. van den Brule, and C. J. Meijer. 2003.
Pathogenic roles for Epstein-Barr virus (EBV) gene products in EBV-associated proliferative disorders. Crit. Rev. Oncol. Hematol. 45:1–36.
23. Middeldorp, J. M., and P. Herbrink. 1988. Epstein-Barr virus specific
marker molecules for early diagnosis of infectious mononucleosis. J. Virol.
Methods 21:133–146.
24. Middeldorp, J. M., and R. H. Meloen. 1988. Epitope-mapping on the Ep-
stein-Barr virus major capsid protein using systematic synthesis of overlapping oligopeptides. J. Virol. Methods 21:147–159.
Modrow, S., et al. 1987. Computer-assisted analysis of envelope protein
sequences of seven human immunodeficiency virus isolates: prediction of
antigenic epitopes in conserved and variable regions. J. Virol. 61:570–578.
Ng, M. H., K. H. Chan, S. P. Ng, and Y. S. Zong. 2006. Epstein-Barr virus
serology in early detection and screening of nasopharyngeal carcinoma. Ai
Zheng 25:250–256.
Ooka, T. 2005. Biological role of the BARF1 gene encoded by Epstein-Barr
virus, p. 613. In E. S. Robertson (ed.), Epstein-Barr virus. Caister Academic
Press, Wymondham, England.
Paramita, D. K., et al. 2007. Native early antigen of Epstein-Barr virus, a
promising antigen for diagnosis of nasopharyngeal carcinoma. J. Med. Virol.
79:1710–1721.
Raab-Traub, N. 2002. Epstein-Barr virus in the pathogenesis of NPC. Semin.
Cancer Biol. 12:431–441.
Sall, A., et al. 2004. Mitogenic activity of Epstein-Barr virus-encoded BARF1
protein. Oncogene 23:4938–4944.
Seto, E., T. Ooka, J. Middeldorp, and K. Takada. 2008. Reconstitution of
nasopharyngeal carcinoma-type EBV infection induces tumorigenicity. Cancer Res. 68:1030–1036.
Seto, E., et al. 2005. Epstein-Barr virus (EBV)-encoded BARF1 gene is
expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression. J. Med. Virol. 76:82–88.
Sheng, W., G. Decaussin, A. Ligout, K. Takada, and T. Ooka. 2003. Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus. J. Virol. 77:3859–
3865.
Stevens, S. J., et al. 2005. Diagnostic value of measuring Epstein-Barr virus
(EBV) DNA load and carcinoma-specific viral mRNA in relation to antiEBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J. Clin. Microbiol. 43:3066–
3073.
Stevens, S. J., et al. 2006. Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load
and carcinoma-specific viral BARF1 mRNA. Int. J. Cancer 119:608–614.
Strockbine, L. D., et al. 1998. The Epstein-Barr virus BARF1 gene encodes
a novel, soluble colony-stimulating factor-1 receptor. J. Virol. 72:4015–4021.
Tanner, J. E., et al. 1997. Antibody and antibody-dependent cellular cytotoxicity responses against the BamHI A rightward open-reading frame-1
protein of Epstein-Barr virus (EBV) in EBV-associated disorders. J. Infect.
Dis. 175:38–46.
Tarbouriech, N., F. Ruggiero, M. de Turenne-Tessier, T. Ooka, and W. P.
Burmeister. 2006. Structure of the Epstein-Barr virus oncogene BARF1. J.
Mol. Biol. 359:667–678.
van Grunsven, W. M., A. Nabbe, and J. M. Middeldorp. 1993. Identification
and molecular characterization of two diagnostically relevant marker proteins of the Epstein-Barr virus capsid antigen complex. J. Med. Virol. 40:
161–169.
van Grunsven, W. M., W. J. Spaan, and J. M. Middeldorp. 1994. Localization and diagnostic application of immunodominant domains of the BFRF3encoded Epstein-Barr virus capsid protein. J. Infect. Dis. 170:13–19.
Verschuuren, E. A., et al. 2002. Treatment of posttransplant lymphoproliferative disease with rituximab: the remission, the relapse, and the complication. Transplantation 73:100–104.
Wang, Q., et al. 2006. Anti-apoptotic role of BARF1 in gastric cancer cells.
Cancer Lett. 238:90–103.
Wang, Y., B. Luo, P. Zhao, and B. H. Huang. 2004. Expression of EpsteinBarr virus genes in EBV-associated gastric carcinoma. Ai Zheng 23:782–787.
Wei, M. X., M. de Turenne-Tessier, G. Decaussin, G. Benet, and T. Ooka.
1997. Establishment of a monkey kidney epithelial cell line with the BARF1
open reading frame from Epstein-Barr virus. Oncogene 14:3073–3081.
Wei, M. X., J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka. 1994.
Expression and tumorigenicity of the Epstein-Barr virus BARF1 gene in
human Louckes B-lymphocyte cell line. Cancer Res. 54:1843–1848.
Young, L. S., et al. 1988. Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J. Gen. Virol. 69:1051–1065.
Young, L. S., and A. B. Rickinson. 2004. Epstein-Barr virus: 40 years on. Nat.
Rev. Cancer 4:757–768.
Yu, M. C., J. H. Ho, S. H. Lai, and B. E. Henderson. 1986. Cantonese-style
salted fish as a cause of nasopharyngeal carcinoma: report of a case-control
study in Hong Kong. Cancer Res. 46:956–961.
Zheng, X., L. Hu, F. Chen, and B. Christensson. 1994. Expression of Ki67
antigen, epidermal growth factor receptor and Epstein-Barr virus-encoded
latent membrane protein (LMP1) in nasopharyngeal carcinoma. Eur. J.
Cancer B Oral Oncol. 30B:290–295.
zur Hausen. A., et al. 2000. Unique transcription pattern of Epstein-Barr
virus (EBV) in EBV-carrying gastric adenocarcinomas: expression of the
transforming BARF1 gene. Cancer Res. 60:2745–2748.